Real-time quantitative PCR experimental scheme optimization.

Because the various experimental scenarios offered by the thermal cyclometers produced by each company are not consistent, the optimization strategies are different, but in each of the scenarios, the factors described above that affect the real-time
PCR
reaction are always connected.
as long as the experimental conditions can be better controlled and the experimental steps optimized, it is not always difficult to obtain satisfactory experimental results. The following article will discuss the optimization of real-time quantitative PCR on some basic parameters and experimental steps that affect the experimental results.. 1. Optimization of basic parameters: concentration of 1.1 MgCl2 In a PCR reaction, the concentration of MgCl2 is essential to affect the activity of the enzyme, not only that, the appropriate concentration of MgCl2 can also get a lower Cp (crossing point) value in the reaction, high fluorescent signal strength and good curve peak. Therefore, the depth of its choice should be careful.
generally speaking, it is
. For PCR reactions with DNA
or
cDNA
as the template, MgCl2 at a concentration of 2-5 mM should be selected, and 4-8 mM should be selected for RT-PCR with mRNA as the template.1.2 template concentration: If the researcher is conducting the first experiment, then a series of dilution concentration templates should be selected to select the most suitable template concentration, if the conditions are difficult, at least two dilution (high and medium, low concentration) should be selected for the experiment.
general, it is appropriate to have Cp in 15-30 cycles, a higher template concentration should be used if it is greater than 30, and a lower template depth should be selected if Cp is less than 15. For the determination of Cp values, the fluorescent signal of the SYBR Green I probe is empirically 2 times higher than that of the background, and the fluorescence intensity of the hybrid probe is 0.3 times higher than that of the background.. 2. Condition optimization when determining DNA using SYBR Green I: concentration of 2.1 MgCl2: Most citation-templates are required for 2-4 mM..2.2 The concentration of the template: The initial experiment requires a series of dilution concentrations, such as conditions, to complete at least two degrees of dilution.
gene
group of DNA between 50 ng-5 pg and prosurgery DNA between about 106 copies..2.3 PCR inhibitors: Usually used to eliminate inhibitors is to dilute the sample, but under certain conditions, the concentration of inhibitors is high, and the amount of templates is small, the dilution method can no longer achieve good results, the reaction sensitivity will be reduced, so the researchers to carry out real-time quantitative PCR research, it is best to choose purified templates..2.4 Concentration of citations: The concentration of citants is a key factor affecting the PCR reaction, the concentration is too low, resulting in incomplete reactions, if there are too many citations, misalmutation and the possibility of producing non-specific products will be greatly increased. For most PCR reactions, 0.5 uM is a suitable concentration, and if the initial selection is not ideal, a selection can be made between 0.3-1.0 uM until a satisfactory result is achieved..2.6 Aeration temperature: The first experimental setting of the aeration temperature should be less than the calculated TM value of 5 degrees C, and then selected within 1-2 degrees C. In general, the aeration temperature is determined by experience, which tends to be significantly different from the calculated TM value.. 3. Condition optimization of RT-PCR with SYBR Green I: concentration of 3.1 MgCl2: different target molecules are selected at different concentrations, usually between 4-8 mM. . 3.2 Template concentration: Rt-PCR experiments can choose between total RNA and mRNA, and the concentration should be between 1 pg-1 ug. For low template concentrations, the appropriate amount of MS2 can be increased or measured with alternated RNA as a vector. . 3.3 Control settings: Each lead should have a negative control, a positive control and a pollution control. . 4. Hybrid probe to determine the concentration of DNA 4.1 MgCl2: 0.5-1.0 mM on top of 2-4 mM, but not more than 2.0 mM. . 4.2 Concentration of hybrid probes: 0.2 uM for each probe in the initial experiment, which can be increased to 0.4 uM if the signal strength does not meet the requirements. . 4.3 Control settings: each quotation should have a negative control, each probe should have a negative control. A positive control is required for each experiment. . 4.4 Other conditions are the same as SYBR Green I. . 5. Quantify RT-PCR in real time with hybrid probes: 5.1 MgCl2 concentration: choose between 4-8 mM. . 5.2 Concentration of hybrid probes: 0.2 uM for initial experiments, which can be increased to 0.4 uM if the fluorescence signal strength is insufficient. . 5.3 Template concentration setting: optimized amplification requires a series of experiments on the degree of knowledge and interpretation, and at least two dilution determinations are required under difficult conditions. A total RNA of 1 pg-1 ug or mRNA is selected, and if the concentration of the template is too small (less than 10 ng/ul), MS2 or alternated RNA can be added as a vector. . 5.4 Control settings: Each lead should be set without template control, positive control and contamination control. . 6. Evaluation of hybrid probes: When experimenting with hybrid probes, care must be taken to prevent the formation of probe-quote djumers and their own extension in the reaction process. The formation of the caustor-probe djumer is mainly due to the fact that the probe can interbreed with the 3' end of the lead, and its formation will cause the djust to amplification, thus competing with the source of the gene of the destination, resulting in a decrease in the efficiency of the reaction.
probe itself can be combined with the gene of interest, and its dechain temperature is higher than the primer, so it may act as a primer and trigger an extended reaction, in order to prevent this phenomenon, usually its 3' end completely
phosphorylation
, so that it can not be extended, if the phosphorylation is incomplete or no phosphorylation, it will produce the by-product of the destination gene, thereby interfering with the experimental results. In view of these two points, the probe should be carefully designed and its end completely phosphate. The current real-time quantitative PCR technology is progressing rapidly, this paper is combined with the work experience of this laboratory and literature, the article mentioned in a series of programs, experimental materials and
instrument equipment
have their own advantages and disadvantages, but also have their own scope of use, can not be generally discussed.
no matter what kind of research researchers want to carry out, they should first find the conditions suitable for their research purposes according to their own circumstances, this paper is only for those who intend to use real-time quantitative PCR to conduct research.
.